Affiliation:
1. Danish Institute for Food and Veterinary Research, DK-1790 Copenhagen V, Denmark
Abstract
ABSTRACT
As part of a large international project for standardization of PCR (Food-PCR;
www.pcr.dk
), a multiplex, multiplatform, ready-to-go enrichment followed by a real-time PCR method, including an internal amplification control, was developed for detection of food-borne thermotolerant campylobacters in chickens. Chicken rinse samples were enriched in Bolton broth for 20 h, a simple and rapid (1-h) resin-based DNA extraction was performed, and DNA samples were then tested with two instrument platforms: ABI-PRISM 7700 and RotorGene 3000. The method was validated against an International Standard Organization (ISO)-based culture method by testing low, medium, and high levels of 12 spiked and 66 unspiked, presumably naturally contaminated, chicken rinse samples. In the RotorGene, a positive PCR response was detected in 40 samples of the 66. This was in complete agreement with the enriched ISO culture. The ABI-PRISM 7700 missed one culture-positive sample. Positive samples contained 10
2
to 10
7
CFU/ml after enrichment in Bolton broth. In the enriched samples a detection probability of 95% was obtained at levels of 1 × 10
3
and 2 × 10
3
CFU/ml in the RotorGene and ABI-PRISM, respectively. The amplification efficiency in both platforms was 90%, although the linear range of amplification of purified genomic DNA was 1.5 × 10
1
to 1 × 10
7
(
R
2
= 1.00) for the RotorGene and 10
3
to 10
7
(
R
2
= 0.99) for the ABI-PRISM. In RotorGene and ABI-PRISM the levels of precision of detection as determined by standard deviation (coefficients of variation) of 6-carboxyfluorescein (FAM) threshold cycle (Ct) values were 0.184 to 0.417 (0.65 to 2.57%) and 0.119 to 0.421 (0.59 to 1.82%), respectively. The results showed a correlation (
R
2
) of 0.94 between the target FAM Ct values and CFU per milliliter of enriched naturally contaminated chicken samples, which indicates PCR's additional potential as a tool for quantitative risk assessment. Signal from the internal amplification control was detected in all culture-negative samples (VIC Ct: 23.1 to 28.1). The method will be taken further and validated in an international collaborative trial with regard to standardization.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Reference20 articles.
1. Anonymous. 2000. Microbiology of food and animal feeding stuffs. Preparation of test samples initial suspension and decimal dilution for microbiological examination. Part 2. Specific rules for the preparation of the initial suspension and decimal dilutions of meat and meat products. International Standard Organisation ISO/CD 6887-2. AFNOR Paris France.
2. Anonymous. 2002. Microbiology of food and animal feeding stuffs. Horizontal method for the detection and enumeration of Campylobacter growing at 41.5°C. Part 1. Detection method. Document ISO/TC 34/SC 9 N 553 revision 2. Result of voting on ISO/CD 10272-1. International Standard Organisation Geneva Switzerland.
3. Anonymous. 2002. Campylobacter. Detection and enumeration of thermotolerant Campylobacter in foods. Draft version. Nordic Committee on Food Analysis Oslo Norway.
4. Anonymous. 2002. Microbiology of food and animal feeding stuffs. Polymerase chain reaction (PCR) for the detection of foodborne pathogens. General method specific requirements. Draft International Standard ISO/DIS 22174. DIN Berlin Germany.
5. Cheng, Z., and M. W. Griffiths. 2003. Rapid detection of Campylobacter jejuni in chicken rinse water by melting peak analysis of amplicons in real-time polymerase chain reaction. J. Food Prot.66:1343-1352.
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